Electronic detection of hybridization on nucleic acid arrays

ABSTRACT

A method of detecting locations on a nucleic acid probe array at which hybridization occurs between targets in a fluid sample and nucleic acid probes disposed on a surface of the nucleic acid probe array, comprising: measuring the temperature at a plurality of locations on the surface of the nucleic acid probe array; applying an oscillating level of energy to the surface of the nucleic acid probe array, thereby causing the temperature at the surface of the nucleic acid probe array to oscillate; and detecting a decreased range of temperature oscillation at at least one of the plurality of locations on the nucleic acid probe array, thereby indicating an increased heat capacity caused by latent heat of hybridization between at least one target in the fluid sample and at least one nucleic acid probe disposed on a surface of the nucleic acid probe array.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application is a regular patent application of andclaims the benefit of priority from U.S. patent application Ser. No.60/126,461 filed Mar. 26, 1999 (Attorney Docket No. 18547-037600), thefull disclosure of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to systems for detectinghybridization between targets in a fluid sample and nucleic acid probesdisposed on the surface of a nucleic acid probe array.

BACKGROUND OF THE INVENTION

[0003] Nucleic acid probe arrays are used for detecting the presence ofvarious target molecules in a fluid sample. The nucleic acid probes arepreferably fabricated directly onto the surface of the probe array usinglight directed synthesis. As the fluid sample is passed over the surfaceof the probe array, the target molecules will hybridize withcorresponding nucleic acid sequences which are attached to the surfaceof the probe array.

[0004] The target molecules are preferably prepared with tags, such asfluorescin, in order to discriminate areas of strong hybridization tothe probes on the array. Specifically, a laser is directed at variousdiscreet locations on the probe array, and fluorescent light is emittedat hybridization locations. By knowing the location of various nucleicacid probe sequences attached to the array (i.e.: when the array wasinitially fabricated), and by determining the locations at whichhybridization has occurred (by detecting fluorescence emissionstherefrom), it is then possible to determine whether various targetmolecules are present in the fluid sample.

[0005] Typically, a microscopic scanning system is used to detect thelocations of hybridization between the target molecules in fluid sampleand the probes on the surface of the probe array. Unfortunately, adisadvantage of optical scanning of the probe array to determinehybridization locations is that it requires a complex scanning andfluorescence detection optical system. As such, precise optics arerequired which are adapted to discriminate between variousmicroscopically small locations on the surface of the probe array.Moreover, a further disadvantage of detecting hybridization by anoptical system is that observation of the behavior of each probe over arange of conditions is often difficult.

SUMMARY OF THE INVENTION

[0006] The present invention provides systems for detecting locations ofhybridization on the surface of a nucleic acid probe array betweentargets present in a fluid sample and nucleic acid probes disposed onthe surface of the probe array.

[0007] In a preferred aspect, the present invention comprises measuringthe temperature at a plurality of discreet locations on the surface ofthe probe array while applying an oscillating level of energy to theprobe array, thereby causing the temperature of the probe array tooscillate.

[0008] By detecting a decreased range of temperature oscillation at atleast one of a plurality of locations on the probe array, hybridizationis detected at the at least one of a plurality of locations on the probearray. Specifically, a decreased range of temperature oscillation at theparticular location on the probe array (in response to a steadyoscillation of energy applied to the array) is indicative of an increasein heat capacity at that location due to the latent heat ofhybridization between at least one least one target in the fluid sample,and at least one nucleic acid probe disposed at the particular locationin question on the surface of the probe array.

[0009] At the point of hybridization between the probe and the targetmolecules, the apparent heat capacity at the probe/target interface willincrease. This increased heat capacity can be sensed as a decreasedoscillating thermal response. Accordingly, by detecting a spike in theapparent heat capacity at the nucleic acid probe/substrate interface(the surface of the probe array), hybridization can be detected.

[0010] In various preferred aspects of the invention, the oscillatinglevel energy applied to the surface of the probe array is applied by aheater disposed under the probe array. Preferably, an array of heatersis disposed under the probe array with each heater being disposed undera small “patch” of probes.

[0011] An advantage of the present invention is that the need foroptical scanning of the probe array to detect hybridization location isovercome. Consequently, the need to preprepare the target molecules witha tag such as fluorescin is also overcome.

[0012] Hybridization bond energies between probes which are attached tothe probe array and target molecules in the fluid sample may varywidely, thus resulting in widely variant equilibrium constants which mayexperience some hysteresis as the stringency (temperature or ionictrough) is varied.

[0013] Accordingly, an optional advantage of using an array of heaterswith each heater being disposed under a patch of probes is that it ispossible to adjust the temperature under each patch of probes to atemperature which is approximately equal to the temperature at whichhybridization occurs between probes at a particular patch of probes andtargets in the fluid sample. An advantage of adjusting the temperatureat each patch of probes (by each heater) is that the probe site can beoptimized for detecting hybridization. This translates to a higherquality signal, at a larger probe range. As such, a further advantage ofthe present system is that it may provide a better signal fordetermining true vs. near matches.

[0014] In preferred aspects of the invention, a temperature monitoringsystem is used to measure the temperature at the plurality of locationson the nucleic acid probe array. This temperature monitoring system maypreferably comprise a differential scanning calorimetry system. Inalternate aspects of the present invention, an infrared scanner may beused to measure the temperature at a plurality of locations on thesurface of the probe array.

[0015] In an exemplary aspect of the invention, the heaters are formedon suspended diaphragms of silicon nitrate and the heaters are made ofpolycrystalline silicon.

[0016] In an alternate embodiment, the nucleic acid probe array isdisposed over an optically absorbing material (for example, a thinnickel film) which is in turn disposed over a thermal insulation layer.In an exemplary aspect of the invention, this thermal insulation layercomprises a material selected from the group consisting of a ceramic,silicon or glass.

[0017] In this alternate embodiment of the present invention, theapplication of an oscillating level of energy to the surface of theprobe array is performed by directing the outputs of first and secondlasers at the surface of the probe array. In this embodiment ofinvention, the first laser is preferably adapted to control the “gross”or a large scale temperature at the probe array with a second laserbeing adapted to “fine tune” the oscillating temperature at the probearray. As such, the second laser acts as a “probe” laser, and aninfrared scanner is preferably used to detect the transient heatingsignal from the probe (i.e.: second) laser.

[0018] In yet another embodiment of the present invention, an electrodeis positioned in the target liquid, an insulating layer is positionedunder the nucleic acid probe array and a silicon n-p-n junction isdisposed underneath the insulating layer.

[0019] In this alternate embodiment of the invention, a laser beam isdirected at the under side of the n-p-n junction, thereby forming acircuit between the n-p-n junction and the electrode in the targetliquid. By measuring the impedance of this circuit, hybridization can bedetected. In preferred aspects of this invention, the laser beam isscanned back and forth across the underside of the n-p-n junction,thereby measuring the impedance of the resulting circuit at a pluralityof discrete locations on the nucleic acid probe array.

[0020] An advantage of this embodiment of the present invention is thatthe laser beam unblocks small localized regions of the n-p-n junction,thus avoiding the problem of parasitic capacitance. As such,hybridization can be detected at small discreet locations on the probearray without interference from hybridization at adjacent locations onthe on the probe array. By avoiding the problem of parasiticcapacitance, a high spatial resolution can be achieved. Specifically,parasitic capacitance is decreased in the present system by the creationof a lightly doped blocking diode region under the nucleic acid probearray and an optical beam (such as a laser beam) is then be used tounblock this region.

[0021] The silicon layer is protected from the target liquid by theinsulating layer. In an exemplary aspect, the insulating layer comprisessilicon nitride, silicon carbide, diamond-like carbon, or boron nitride.

[0022] A depletion region will form in the silicon layer adjacent to theinsulating layer. The n-p-n junction forms an additional depletionregion which reduces the parasitic capacitance.

[0023] Moreover, the AC impedance of the circuit will be sensitive tochanges close to the insulating layer within a distance defined as theDebye layer. By scanning the laser light beam across the underside ofthe system, a local hybridization pattern can be determined.

[0024] In accordance with this embodiment of the present invention,hybridization locations may be detected electronically by: 1) detectinga shift in double-layer capacitance resulting from a change in thedielectric constant, 2) detecting a shift in the point of zero charge ina semiconductor electrolyte interface, and 3) detecting a shift in thezeta potential of the insulating layer. The second and third mechanismspreferably require a semiconductor substrate.

[0025] In various alternate embodiments, the p-n junction is eliminated;photocurrent is used to measure surface changes, an ion sensitivematerial is placed over the insulating layer; and/or the device may bedefined on an insulator using SOI technology to reduce parasiticcapacitance.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a sectional elevation view of a first embodiment of thepresent invention.

[0027]FIG. 2 is a top schematic view of an array of heaters disposedunder “patches” of nucleic acid probes on a nucleic acid probe array.

[0028]FIG. 3 is a sectional elevation view of a second embodiment of thepresent invention.

[0029]FIG. 4 is a sectional elevation view of a third embodiment of thepresent invention.

[0030]FIG. 5 is a graph showing the relationship over time between themeasured temperature and the energy applied to a location on the probearray at which hybridization occurs.

[0031] Lastly, FIG. 6 shows the relationship between time andtemperature at a location on the probe array at which hybridization hastaken place, in the case where the energy applied to the probe array iscontinuously increased over time.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0032] Referring first to FIG. 1, a first embodiment of the presentinvention is provided. A nucleic acid probe array 10 is provided havinga plurality of nucleic acid sequences (i.e.: probes) 12 attachedthereto. A plurality of target molecules 14 (freely floating within afluid sample) are passed over the surface of array 10. When a targetmolecule (14A) corresponds identically to an opposite probe sequence(12A) hybridization will occur, as shown.

[0033] The present invention provides systems for detectinghybridization (for example between probe 12A and target molecule 14A) bydetecting the location on array 10 at which such hybridization hasoccurred. Since the identity and location of probe 12A is known whenarray 10 is initially fabricated, the presence of target molecule 14A inthe fluid sample can be confirmed simply by detecting hybridization atthe location occupied by probe 12A on array 10.

[0034] In a first embodiment of the present invention, a plurality ofheaters 20 are disposed underneath the probes 12 on array 10, as shown.

[0035] In accordance with the present invention, the temperature ismeasured at various discreet locations on the probe array while anoscillating (or otherwise varying) level of energy is applied to theprobe array, for example by heating heaters 20.

[0036] In one aspect of the invention the temperature of probe array 10is oscillated and hybridization is detected by sensing a decrease in therange of temperature oscillation at the location of hybridization,(which is caused by an increased heat capacity caused by the latent heatof hybridization between the probe and the target molecule).

[0037] In an alternate approach, the energy applied to probe array 10 isvaried (e.g.: increased), with continuous temperature monitoring showinga decrease or “dip” in the rate of increase of the temperature at thatlocation when hybridization occurs. Conversely, the temperature of probearray 10 may be decreased, (by allowing the array to cool) with adecrease or “dip” in the rate of decrease of the temperature at alocation on the probe array also indicating that hybridization hasoccurred.

[0038] As show in FIG. 1, each of heaters 20 may be disposed under a“patch” of probes. In a preferred aspect, heaters 20 are each producedto be very small such that each resides under a minimum number of probes12. As such, it is possible to operate each of heaters 20 such that thetemperature of the probe(s) disposed over the heater can be as close aspossible to the temperature at which hybridization actually occursbetween a particular probe(s) and it's corresponding target sequence(s).Having a plurality of heaters 20 disposed in an array under patches ofprobes (see FIG. 2) allows different portions of array 10 to be adjustedto different temperatures, thereby enhancing signal results.

[0039]FIG. 2 shows a second embodiment of the present invention in whichenergy is applied to the surface of probe array 10 by a first laser 30and a second laser 32. In an exemplary aspect, first laser 30 controlsthe “gross” temperature at the surface of array 10 and second laser 32is operated to “fine tune” the temperature at the surface of array 10.An infrared scanner 40 may be used to measure the temperature at each ofthe plurality of discreet locations on array 10, with the temperatureresponse measured at each of these locations being used to determinewhether hybridization has occurred, using any of the methods asexplained above.

[0040] In this second embodiment, a light absorbing layer 50, a thermalinsulation layer 52 and a ceramic, silicon or glass substrate 54 may bepositioned under array 10 as shown.

[0041] In a third embodiment of the present invention, as shown in FIG.4, an electrode 60 is positioned in the target liquid which is passedover the surface of array 10. An insulating layer 62 is disposed underarray 10 and an n-p-n junction (formed from n-type doped silicon layer64, p-type doped silicon layer 66, and n-type doped silicon layer 68) isdisposed under insulating layer 62, as shown.

[0042] A laser 70 scans a laser beam 72 across the underside of then-p-n junction, forming a circuit between the n-p-n junction andelectrode 60. By measuring the impedance of this circuit (with only thelocation at which laser beam 72 strikes the underside of the n-p-njunction being unblocked), the pattern of hybridization can be detectedacross array 10.

[0043]FIG. 5 illustrates the relationship over time between the measuredtemperature and the energy applied to a location on the probe array atwhich hybridization occurs, with the point at which hybridization occursbeing shown as a decrease in the temperature relative to the amount ofenergy applied to the surface of the probe array.

[0044] Lastly, FIG. 6 shows the relationship over time of thetemperature of the probe array at a location on the probe array at whichhybridization has taken place, in the case where the energy applied tothe probe array is continuously increased over time

What is claimed is:
 1. A method of detecting locations on a nucleic acidprobe array at which hybridization occurs between targets in a fluidsample and nucleic acid probes disposed on a surface of the nucleic acidprobe array, comprising: measuring the temperature at a plurality oflocations on the surface of the nucleic acid probe array; applying anoscillating level of energy to the surface of the nucleic acid probearray, thereby causing the temperature at the surface of the nucleicacid probe array to oscillate; and detecting a decreased range oftemperature oscillation at at least one of the plurality of locations onthe nucleic acid probe array, thereby indicating an increased heatcapacity caused by latent heat of hybridization between at least onetarget in the fluid sample and at least one nucleic acid probe disposedon a surface of the nucleic acid probe array.
 2. A method of detectinglocations on a nucleic acid probe array at which hybridization occursbetween targets in a fluid sample and nucleic acid probes disposed on asurface of the nucleic acid probe array, comprising: measuring thetemperature at a plurality of locations on the surface of the nucleicacid probe array; applying energy to the surface of the nucleic acidprobe array, thereby causing the temperature at the surface of thenucleic acid probe array to increase; and detecting a decrease in therate of temperature change at at least one of the plurality of locationson the nucleic acid probe array, thereby indicating an increased heatcapacity caused by latent heat of hybridization between at least onetarget in the fluid sample and at least one nucleic acid probe disposedon a surface of the nucleic acid probe array.
 3. A method of detectinglocations on a nucleic acid probe array at which hybridization occursbetween targets in a fluid sample and nucleic acid probes disposed on asurface of the nucleic acid probe array, comprising: measuring thetemperature at a plurality of locations on the surface of the nucleicacid probe array; decreasing the temperature of the surface of thenucleic acid probe array; and detecting a decrease in the rate oftemperature change at at least one of the plurality of locations on thenucleic acid probe array, thereby indicating an increased heat capacitycaused by latent heat of hybridization between at least one target inthe fluid sample and at least one nucleic acid probe disposed on asurface of the nucleic acid probe array.
 4. The method of claim 1 or 2,wherein applying energy to the surface of the nucleic acid probe arrayis accomplished by at least one heater disposed under the nucleic acidprobe array.
 5. The method of claim 4, wherein the at least one heatercomprises an array of heaters, each heater being disposed under a patchof probes.
 6. The method of claim 1, wherein the at least one heatercomprises an array of heaters, each heater being disposed under a patchof probes, further comprising: adjusting the temperature at each patchof probes to a temperature approximately equal to the temperature atwhich hybridization occurs between the patch of probes and the targets.7. The method of claim 1, further comprising: providing an opticallyabsorbing layer under the probe array; providing a thermal insulationlayer under the optically absorbing material; and providing a substrateunder the thermal insulation layer.
 8. The method of claim 7, whereinthe substrate comprises a material selected from the group consisting ofa ceramic, silicon or glass.
 9. The method of claim 1 or 2, whereinapplying an oscillating or varying level of energy to the surface of thenucleic acid probe array comprises: directing the output of a firstlaser at the surface of the nucleic acid probe array.
 10. The method ofclaim 9, wherein applying an oscillating or varying level of energy tothe surface of the nucleic acid probe array further comprises: directingthe output of a second laser at the surface of the nucleic acid probearray, wherein the output of the first laser is greater than the outputof the second laser.
 11. The method of claim 10, wherein the first laseris adapted to control the average temperature at the probe array, andthe second laser is adapted to fine tune the temperature at the probearray.
 12. The method of claim 7, wherein the optically absorbing layercomprises a thin nickel film.
 13. The method of claim 7, wherein aninfrared scanner is used to measure the temperature at a plurality oflocations on the surface of the nucleic acid probe array.
 14. A systemfor detecting locations on a nucleic acid probe array at whichhybridization occurs between targets in a fluid sample and nucleic acidprobes disposed on a surface of the nucleic acid probe array,comprising: a nucleic acid probe array; at least one heater disposedunder the nucleic acid probe array; and a temperature monitoring systemfor measuring the temperature at a plurality of locations on the nucleicacid probe array.
 15. The system of claim 14, wherein the at least oneheater comprises an array of heaters.
 16. The system of claim 14,wherein the temperature monitoring system comprises: an infraredscanning system.
 17. The system of claim 14, wherein the heaters aremade of polycrystalline silicon formed on suspended diaphragms ofsilicon nitrate.
 18. The system of claim 14, further comprising: anoptically absorbing layer disposed under the probe array; a thermalinsulation layer disposed under the optically absorbing material; and asubstrate disposed under the thermal insulation layer.
 19. A method ofdetecting locations on a nucleic acid probe array at which hybridizationoccurs between targets in a fluid sample and nucleic acid probesdisposed on a surface of the nucleic acid probe array, comprising:positioning an electrode in the target liquid; providing an insulatinglayer under the nucleic acid array; providing a n-p-n junction under theinsulating layer; directing a laser beam at the underside of the n-p-njunction, thereby forming a circuit between the n-p-n junction and theelectrode in the target liquid; and measuring the impedance of thecircuit.
 20. The method of claim 19, further comprising: scanning thelaser beam across the underside of the n-p-n junction, thereby measuringthe impedance of the circuit at a plurality of locations on the nucleicacid array.
 21. The method of claim 19, wherein the insulating layercomprises silicon nitride.
 22. A system for detecting locations on anucleic acid probe array at which hybridization occurs between targetsin a fluid sample and nucleic acid probes disposed on a surface of thenucleic acid probe array, comprising: an electrode adapted to bedisposed in the target liquid; an insulating layer disposed under thenucleic acid array; and a n-p-n junction disposed under the insulatinglayer.
 23. The system of claim 22, further comprising: a laser adaptedto direct a laser beam at the underside of the n-p-n junction, therebyforming a circuit between the n-p-n junction and the electrode in thetarget liquid.